Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Note: All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center San Antonio and were consistent with the ARRIVE guidelines.
1.&#160;Anesthetizing for Cortical Preparation
<ol>
	<li>Place the mouse in an induction chamber with 2-3% isofluorane mixed with oxygen to induce anesthesia. Observe the respiration rate decrease as the mouse is induced. Pinch the paw of the mouse to determine whether the mouse is ready to mo...

<ol>
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M., Wewer, J., Lechleiter, J. P. 2. Y. 1. R. -. i. n. i. t. i. a. t. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IP3R-dependent+stimulation+of+astrocyte+mitochondrial+metabolism+reduces+and+partially+reverses+ischemic+neuronal+damage+in+mouse.">IP3R-dependent stimulation of astrocyte mitochondrial metabolism reduces and partially reverses ischemic neuronal damage in mouse.</a> Journal of Cerebral Blood Flow and Metabolism. 33, 600-611 (2013).</li><li>Witte, O. 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Y., Rafie, C., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topological+basis+for+the+robust+distribution+of+blood+to+rodent+neocortex.">Topological basis for the robust distribution of blood to rodent neocortex.</a> Proceedings of the National Academy of Sciences. 107, 12670-12675 (2010).</li><li>Nishimura, N., Rosidi, N. L., Iadecola, C., Schaffer, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+collateral+flow+after+occlusion+of+a+single+cortical+penetrating+arteriole.">Limitations of collateral flow after occlusion of a single cortical penetrating arteriole.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 30, 1914-1927 (2010).</li><li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. 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Rinsho shinkeigaku.</a> Clinical Neurology. 50, 455-460 (2010).</li><li>Tiannan Wang, W. C., Xie, Y., Zhang, W., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+Volume+of+the+Focal+Cerebral+Ischemic+Lesion+through+Photothrombosis.">Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis.</a> American Journal of Biomedical Sciences. 2, 33-42 (2009).</li><li>Head, B. P., Patel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+brain+protection.">Anesthetics and brain protection.</a> Current Opinion in Anaesthesiology. 20, 395-399 (2007).</li><li>Kirsch, J. R., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+and+cerebroprotection:+experimental+aspects.">Anesthetics and cerebroprotection: experimental aspects.</a> International Anesthesiology Clinics. 34, 73-93 (1996).</li><li>Koerner, I. P., Brambrink, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+protection+by+anesthetic+agents.">Brain protection by anesthetic agents.</a> Current Opinion in Anaesthesiology. 19, 481-486 (2006).</li><li>Gelb, A. W., Bayona, N. A., Wilson, J. X., Cechetto, D. 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The ability to translate experimental stroke pathophysiology from animal to human application has been plagued with failure. However, the use of animal models, such as the photothrombosis model, allows for improved understanding of stroke pathophysiology and the exploration of new therapeutic approaches to provide neuroprotection following a stroke. Small cortical strokes and microinfarctions produced by the photothrombotic model are clinically relevant to subclinical or &#8220;silent&#8221; stroke13-15, which...

The technique described permits visualization of in vivo cellular responses immediately following induction of Rose Bengal photothrombosis in an intact mouse. Rose Bengal (4,5,6,7-tetrachloro-2&#39;,4&#39;,5&#39;,7&#39;-tetraiodofluorescein) is a photosensitive dye used to induce ischemic stroke in animal models (mouse and rat). Following a bolus injection of RB through the tail vein and subsequent illumination through a thinned skull with a 564 nm laser light, a thrombus is induced causing a physiologic stroke1. The method was originally described by Rosenblum and El-Sabban in 1977, and was later adapted by Watson in the mid 1980s1,2. In brief, Rose Bengal is irradiated with green excitation light (561 nm laser in our case), which generates the production of reactive oxygen species, which subsequently activates tissue factor, an initiator of the coagulation cascade. The induction of the coagulation cascade produces an ischemic lesion that is pathologically relevant to clinical stroke3.
Stroke has a complex pathophysiology due to the interplay of many different cells types including neurons, glia, endothelium and the immune system. Choosing the best technique to study a particular cellular process requires multiple considerations. Experimental techniques fall broadly into one of three categories: in vitro, in vivo and in silico with each having advantages and disadvantages. In vitro studies have the primary disadvantage of removing cells from their natural environment and therefore may not reproduce effects seen in an intact, living animal. In vivo techniques provide for enhanced experimental replication of disease states with increased translational significance. In silico generally refers to computer modeling of a disease or cellular process, and while increasingly utilized to study potential drug interactions for example, any information gleaned must still be tested in living cells or tissue.
The ideal model of stroke in the laboratory setting should demonstrate similar pathological features to those seen in the human population. While there are common physiologic characteristics of stroke in the human population, there are also many differences depending on the type of injury experienced. Stroke in the human population occurs as small or large vessel occlusions, hemorrhagic lesions, and artery-to artery or cardio-embolisms that result in varied infarct volumes as well as differences in mechanisms related to each pathology. The advantage of utilizing animal stroke models is the generation of reproducible infarcts that mimic characteristics of human stroke. The most common animal stroke models include artery occlusion using: middle cerebral artery occlusion (embolic or endovascular filament methods) which models distal MCAO and the photothrombosis model. The advantages and disadvantages of each model have been reviewed elsewhere (see 4 and 5). Global ischemic models (MCAO), while relatively easy to perform are less relevant to human stroke than are focal stroke models. In addition, these methods are highly variable in inducing reproducible brain infarct lesions. The photothrombosis model is highly reproducible as long as the experimenter controls their experiments well, providing a clear advantage over MCAO models. However, due to microvasculature insult the model has been described to display a minimal ischemic penumbra, the area where cells are thought to be salvageable 6,7. Additionally, vasogenic edema and cytotoxic edema formation may also be induced following irradiation of the imaging area. Despite these limitations the technique has provided new insight into many physiological processes following stroke8,9,10,11.

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			<td>Reagents</td>
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			<td>Rose Bengal</td>
			<td>Sigma</td>
			<td>330000</td>
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			<td>aCSF&#160;</td>
			<td>126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 26 mM NaHCO3 (pH 7.4).</td>
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			<td>[header]</td>
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			<td>123-6670</td>
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			<td>Optosigma Co</td>
			<td>145-1110</td>
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			<td>Ear bar holder from stereotaxic setup</td>
			<td>Stoelting/Cyborg</td>
			<td>51654</td>
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			<td>Dispomed Labvent Rodent anesthesia machine</td>
			<td>DRE, Inc.</td>
			<td>15001</td>
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			<td>Tech IV Isoflurane vaporizer</td>
			<td>DRE, Inc.</td>
			<td>34001</td>
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			<td>F Air Canister</td>
			<td>DRE, Inc</td>
			<td>80120</td>
			<td></td>
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			<td>Bain circuit breathing tube</td>
			<td>DRE, Inc</td>
			<td>86111B</td>
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			<td>Rodent adapter for bain tube</td>
			<td>DRE, Inc</td>
			<td>891000</td>
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			<td>DRE, Inc</td>
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			<td>Foredom drill Dual voltage 110/120</td>
			<td>Foredom</td>
			<td>134.53</td>
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rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

The aim of this method was to induce an ischemic stroke in animal models (mouse and rat) following a bolus injection of RB through the tail vein and subsequent illumination of a thinned skull with a 561 nm laser light. The images in Figure 4 demonstrate the progression of clot formation within a single vessel following irradiation of the area at 0, 1, 1.5 and 2 min. Prior to clot formation the entire vessel is white due to free flowing Rose Bengal. Following the induction of irradiation of the vessel the...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

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